African Journal of Biotechnology Vol. 9(42), pp.

7108-7113, 18 October, 2010

Available online at http://www.academicjournals.org/AJB
ISSN 16845315 2010 Academic Journals



Full Length Research Paper

Effects of fractionation on antibacterial activity of crude
extracts of Tamarindus indica

U. U. Nwodo
1
*, A. A. Ngene
2
, C. U. Iroegbu
1
and Obiiyeke GC
3


1
Department of Microbiology, University of Nigeria, Nsukka, Enugu State, Nigeria.
2
Department of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria.
3
Department of Botany, Delta State University, Delta State, Nigeria.

Accepted 26 February, 2010

Column chromatographic fractionation of the crude ethanolic extract of the stem bark of Tamarindus
indica yielded six fractions (TiA - TiF). Among these, TiB showed about five tracks: TiC, TiD and TiE,
two tracks each, on thin layer chromatography (TLC). Fractions TiC, TiD and TiE were re-eluted with
different solvent systems and each yielded two sub-fractions, while fraction TiB yielded four. All
fractions and sub-fractions tested for antibacterial activity in vitro using the agar well diffusion
technique. TiA showed activity against 100% of the test gram negative bacterial strains and 60% of the
gram positive strains; TiB, TiC, and TiD each showed activity against 71.4% of the gram negative test
strains and 100, 80 and 60%, respectively, of the gram positive strains. Fractions TiE and TiF,
respectively, showed activity against 42.9 and 14.3% of the gram negatives and 60 and 20% against the
gram positives bacteria. The crude extract and Ciprofloxacin (control), respectively, were active against
57.1 and 100% of the gram negatives; and 80 and 100% of the gram positives. The activities of the sub-
fractions of TiB, TiC, TiD and TiE against the test strains varied from those of the parent fractions. The
phytochemistry of these fractions showed varied contents of tannins, saponins, flavonoids, alkaloids,
antroquinone, glycosides and terpene.

Key words: Fractionation, chromatography, plant extract, Tamarindus indica, antibacterial activity,
phytochemistry.


INTRODUCTION

Plants produce a good deal of secondary metabolites
which have benefited mankind in various ways including
treatment of diseases (Elaine et al., 2002). These meta-
bolites serve different purposes in the plant, including
growth regulation, allelopath, defense against predators
and infections or they may be waste products. Outside
their intrinsic uses in the plant, these secondary meta-
bolites have variously been shown to exhibit interesting
biological and pharmacological activities and are important
as prophylactics, chemotherapeutics or have served as
the starting points in the development of modern medicines
(Verpoorte, 1998). Thus, a crude plant Extract is a
complex mixture and its evaluation for the large array of
compounds in the complex mixture may interact anta-



*Corresponding author. E-mail: uchenwodo@gmail.com.

Abbreviations: TLC, Thin layer chromatography; CFU, colony
forming unit; IZD, inhibition zone diameters.
gonistically interfering with or masking the activity of one
another. Secondly, the vast majority of active compounds
in crude extracts is present at a very low concentration
and therefore may not show high specific activity.
One approach to solving these problems has been to
separate the compounds to greater purity and to con-
centrate them by various processes, including by chroma-
tography (Jean et al., 2001). It is not always, however, that
fractionation of crude extracts improves activity in
spectrum and or in specificity and intensity. The effica-
cious use of crude extracts or concoctions by herbal
healers immediately suggests activity of components
singly or synergistically in combination. Besides, com-
binations of these components in nature may interact to
reduce toxicity. Hence, purification of crude extracts to
concentrate the active principles in line with modern
pharmacological practice is thought to result, sometimes,
in loss of activity and/or increase in toxicity.
Tamarindus indica L., (Tamarind), family, Leguminosae, is
widely used as both food and medicine in many West
African communities (Anon, 1986; Morton, 1987). The pulp




extracts has been documented as antipyretic, antiscorbutic,
laxative, carminative and remedy for biliousness and bile
disorder (Raimondi, et al., 2003; Morton, 1987; Iwu,
1993). Furthermore, extracts of various parts of the plant
have been reported to have antibacterial properties
(Doughari, 2006). This study was undertaken, therefore,
to fractionate crude ethanolic stem bark extracts of T.
indica by column chromatography and evaluate the
fractions for antibacterial activity as required for inclusion
of herbal medicines in orthodox medical practice.


MATERIALS AND METHODS

Plant materials

The stem bark of T. indica plant evaluated was obtained from More,
Sokoto South Local Government Area, Sokoto State, Nigeria. The
plant was taxonomically identified at the Department of Botany,
University of Nigeria, Nsukka and a voucher specimen deposited in
the herbarium of the Department.


Extractions of the plant materials

Fresh stem bark of T. indica were rinsed thoroughly in running tap
water, chopped to tiny pieces and air dried in the dark at room
temperature (~ 28C). The dried stem bark was pulverised into
powder using a mechanical hammer mill. A 50.0 g weight of the
milled material was macerated into 200 ml of absolute ethanol
(Sigma-Aldrich) and left to stand for about four (4) hours. The
preparation was filtered through Whatman No. 1 paper and filtrate
concentrated to dryness in a steady air current. The extract was
stored in sterile containers at a temperature of 4C until further
used.


Fractionation of extract

The crude ethanolic extract of the stem bark of T. indica was re-
constituted in absolute ethanol and spotted on analytical TLC (silica
gel G600, 0.25 mm thickness) and the following solvent systems and
ratios used as mobile phase to determine the eluent with optimum
performance; benzene/ethyl acetate 9:1, 8:2, 7:3, 6:4, and 1:1;
ethanol/ethyl acetate 9:1, 8:2, 7:3, 6:4 and 1:1; methanol/ ammonia/
water 8:1:1, chloroform/ethanol 9:1, chloroform/ethanol 1:1 and
dichloromethane. After each separation, the TLC plate was
exposed to iodine fumes in a chamber. The solvent system giving
the best resolution was adopted for column fractionation. Column
separation of the extracts was carried out with a glass column of
internal diameter 80 mm and length 100 cm (Quickfit, England).
Sufficient quantity of a column grade silica gel (120 - 200 mesh
size) was wet-packed using benzene/ ethylacetate (6:4) solvent
system. A 40 g amount of the crude extract was first dissolved in 20
ml of ethanol, and then mixed with about 20 g of the silica gel to
become slurry. The slurry was loaded onto the wet packed column
and continuously eluted with the mobile phase (ethanol/ ethylacetate,
6:4). Approximately 20 ml aliquots of eluent were collected while
observing the distance traveled by the sample down the column; in
addition, bands of the same sample formed on TLC were also
monitored. The fractions showing similar TLC mobility and band
formation were pooled and the solvent evaporated under a steady
air current at room temperature. Fractions which did not give single
sharp band on the TLC were re-fractionated using the same silica
gel column but different solvent system. The solvent systems
employed for this refraction were benzene/ethylacetate (6:4), ethanol/
Nwodo et al. 7109



ethylacetate (6:4), dichloromethane and methanol.


Phytochemical screening

The crude extract, fractions (TiA - TiF) and sub-fractions from re-
fractionated fractions were screened for the presence of alkaloids,
saponins, tannins, anthraquinones, glycosides, flavonoids, reducing
sugar, carbohydrates and sterols using standard phytochemical
methods (Trease and Evans, 1978; Harbone, 1998).


Test bacterial strains

Clinical isolates of Staphylococcus aureus from a case non-
gonococcal urethritis and Escherichia coli from a case of
gastroenteritis; and two strains of typed Bacillus cereus (NRRL
14724 and NRRL 14725) were collected from the Clinical Diagnostic
Laboratory, Department of Microbiology, University of Nigeria,
Nsukka. Typed strains of Pseudomonas aeruginosa (ATCC 10145),
E. coli (ATCC 11775), Bacillus subtilis (ATCC 6051), and S. aureus
(ATCC 12600) were obtained from Bioresources Development and
Conservation Project (BDCP), Nsukka. Salmonella kintambo (SSRL
113) was supplied by the Veterinary Microbiology and Pathology
Laboratory of the University of Nigeria, Nsukka. Each test bacterial
strain was purified by re-isolating severally on Mueller Hinton agar
(Oxoid) and emergent discrete colonies picked and identity
reaffirmed after characterization by standard bacteriological method
(Cheesbrough, 1984). Stock cultures were maintained in nutrient
agar slants at +4C.


Assaying extracts for antibacterial activity

Fractions were assayed for antibacterial activity using the agar well
diffusion technique. Inoculum of test bacterial was standardized by
McFarland Nephelometry (NCCLS M2-A5, 1993); thereafter, Gram-
positive bacteria were adjusted to 1.0 x 10
6
CFU/ml and gram-
negative bacteria to 5 x 10
5
CFU/ml (NCCLS M2-A5, 1993). A 100
l volume of the standardized test bacterial suspension was seeded
and spread evenly on to each sterile Muller Hinton agar plate so as
to achieve a confluent growth. The plates were allowed to dry and a
sterile 6.0 mm-diameter cork borer was used to drill wells in the
agar plates. The extracts were reconstituted with sterile distilled
water to a concentration of 62.5 mg/ml; and 100 l introduced in
triplicate wells in the MHA cultures. The plates were allowed to
stand for 2 h at room temperature for diffusion to take place and
then incubated at 37C for 24 h. The inhibition zone diameter was
measured to the nearest mm.


RESULTS

Column chromatography of the crude ethanolic extract of
T. indica stem bark yielded six fractions designated TiA,
TiB, TiC, TiD, TiE and TiF (Table 1). On subsequent
subjection of the fractions to TLC, TiA and TiF, each
showed a single band whereas TiB, TiC, TiD and TiE
showed 5, 3, 2 and 2 bands, respectively. The pH and
yield of the stem bark crude ethanolic extracts and
fractions of T. indica is shown in Table 2. Fraction TiB
had more yield (12.0%) than others and TiF, the least
(4.5%). The pH values of both crude and fractions ranged
from 3.74 (fraction TiA) to 4.61 (crude extract). On re-
fractionation, TiB yielded further four distinct fractions
7110 Afr. J. Biotechnol.



Table 1. Chromatographic separations of the stem bark of Tamarindus indica.

Sample
Products of first
fractionation
Tracks on TLC Products of re-fraction Tracks on TLC
TiA 1 - -
TiB 5 B1, B2, B3, and B4 1
TiC 3 C1 and C2 1
TiD 2 D1 and D2 1
TiE 2 E1 and E2 1
Crude
ethanol
extract of
stem bark

TiF 1 - -



Table 2. The yield and pH of crude extract of Tamarindus indica and
its fractions.

Fractions pH Yield % yield
Crude 4.61 4.50 9.0
TiA 3.74 2.3 5.79
TiB 4.21 4.8 12.0
TiC 4.18 3.6 9.0
TiD 4.21 3.9 9.75
TiE 4.43 2.4 6.0
TiF 4.32 1.8 4.50



Table 3. Phytochemical composition of initial fractions of crude extract of the stem bark of
Tamarindus indica.

Constituents TiA TiB TiC TiD TiE TiF Crude
Carbohydrates - - + + + + + + + + +
Reducing sugar - - - + + + +
Tannins + + + - - - - +
Flavonoids + + + + + + + + + + + + +
Anthroquinone - + + + + - - - + +
Saponins + + + + + + + + + + + + + - + + +
Alkaloids - + + + + + + - + + +
Cyanogenic glycoside - + + + - - - + +
Terpenes - - + - - - -
Sterol - - - - - + -

- = Not detectable; + = present in trace quantity; + + = moderately present; + + + = highly present.



(B1, B2, B3 and B4) which expressed single track on TLC
each; the others yielded two further fractions each,
namely, TiC C1, C2), TiD (D1, D2) and TiE (E1, E2)
(Table 1). The phytochemical analysis showed that TiA,
TiB, TiD and TiE contained saponins, flavonoids and
alkaloids. Tannins were only detected in TiA and TiB;
anthroquinone, terpene and cyanogenic glycosides only
in TiC and Sterol only in TiF (Table 3). Sub-fractions of
TiB1, TiB2, TiB3, TiB4, TiC1 and TiC2 all contained saponin
while alkaloids were detected in TiB4, cyanogenic glycol-
sides in TiB3, Terpene in TiB1 and sterol in TiE1 and
TiE2 (Table 4).
All six parent fractions TiA to TiF from the crude extract
showed antibacterial activity with IZDs (inhibition zone
diameters) ranges of 9.0 1.42 mm against S. kintambo
SRRL; 113 to 13.0 0.5 mm against B. cereus NRRL;
14724 for TiA; 9.50 0.25 mm against Proteus mirabilis
(clin.) to 17.0 0.71 mm against P. aeruginosa; ATCC
10145 for TiB; 9.0 1.2 mm against S. aureus ATCC;
12600 to 15.0 0.25 mm against E. coli (clin.) for TiC;
9.0 1.2 mm against S. aureus (clin.) to 15.0 0.25 mm
against E. coli; ATCC 11775 for TiD; 10.0 mm against S.
aureus ATCC 12600 to B. subtilis; ATCC 6051 for TiE;
and 10.0 0.25 mm against P. aeruginosa ATCC; and
10145 to 12.0 0.25mm against S. aureus for TiF (Table
5). Sub-fractions TiB1, TiB3, TiB2 and TiB4, respectively,
Nwodo et al. 7111



Table 4. Phytochemical composition of re-fractions.

TiB TiC TiD TiE
Phytoconstituent
B1 B2 B3 B4 C1 C2 D1 D2 E1 E2
Carbohydrates - - - - + + + + + + + - + +
Reducing sugar - - - - + + + + - +
Tannins - + + - + - + - - - -
Flavonoids - + + - - - + + + - + - +
Anthroquinone - + - + - + - - - -
Saponins + + + + + + + + + + + + + - -
Alkaloids - + + + + + - + - + - -
Cyanogenic
glycoside
- + + + + - - + - - - -
Terpenes + + + - - + + + + - - - -
Sterol - - - - - - - - + +

- = Not detectable; + = present in trace quantity; + + = moderately present; + + + = highly present.



Table 5. The antibacterial activities of the fractions and crude ethanolic extract of Tamarindus indica stem bark.

Hean |nh|b|t|on zone d|ameter [125 mg|m|}
acter|a| stra|n
6rude
extract [sbf}
T| [A} T| [} T| [6} T| [0} T| [E} T| [F}
6|prof|ox [20
g|m|}
E. co|| 20.50 0.Z1 11 0.25 1 0.Z1 1 0.Z1 15 1.2 13 0.25 0 2.25 0.25
E. co|| ATCC 11ZZ5 8.50 0.Z1 12 0.Z1 15.5 0.Z1 15.5 0.Z1 15 0.25 0 0 32.85 0.25
$a|mone||a ryon| 0 11 0.5 0 0 0 0 0 21.0 0.25
$a|mone||a k|nramoo
33RL 113
0 9 1.12 0 11 1.0 12 0.25 10 0.25 0 21.80 0.0
$raon||ococcus aureus 11.50 0.Z1 0 1.50 0.25 9 0.Z1 9 1.2 11 1.20 12 0.25 25.85 0.25
$raon. aureus ATCC
1200
0 10.50 0.50 12 0.Z1 9 1.2 0 10 0.0 0 22.25 0.15
Pseuoomonas
aeruo|nosa
21.0 1.21 10 0.25 13.50 0.Z1 8 8.50 0.25 0 0 25.85 0.25
Pseuoomonas
aeruo|nosa ATCC
10115
1Z.0 0.0 12.50 0.5 1Z 0.Z1 11 1.2 11 0.25 12 0.0 10 0.25

23.25 0.25
3. suor|||s ATCC 051 12.50 0.Z1 11 1.12 12 0.50 11.50 1.0 0 11 0.25 0 31.0 0.Z5
Proreus m|rao|||s 15.0 0.0 10 0.25 9.50 0.25 0 0 0 0 21.0 1.0
3. cereus NRRL 11Z21 15.0 0.0 13 0.5 1.50 2.1 0 11 0.25 0 0 2Z.25 0.25
3. cereus NRRL 11Z25 10.0 1.11 0 13 0.Z1 11 0.5 10 0.25 0 0 2.0 1.00

Sbf = Stem bark fraction; ciproflox = ciprofloxacin antibiotic.



showed activity against 57.0, 42.9, 28.6 and 14.2% of the
test gram negative test bacterial strains compared with
the 71.43% spectrum of activity of the parent fraction
(Figure 1). Similarly, TiB1and TiB2 were active against
20% and TiB3 and TiB4 against 60 and 40%, respec-
tively, of the gram positive test bacterial strains (Figure
2). Sub-fraction TiC1 was active against all the gram
negative strains and 60% of the gram positive strains;
TiC2 showed activity against 42.9% of the gram negative
strains and 40% of the gram positive strains. Also, TiD2
showed activity against 71.40% of the gram negative
bacterial strains and 60% of the gram positives; TiE1
showed activity against 57.10% of the gram negatives
and 40% of the gram positives but TiE2 showed activity
against 41.86% of the gram negatives and 60% of the
gram positives, respectively.


DISCUSSION

All the fractions and sub-fractions had acid pH (3.74 -
4.61) which means that the aggregate hydrogen ion (H
+
)
effect of the component compounds is acidic and
consistent with the reported pH of extracts from most
7112 Afr. J. Biotechnol.



0
5
10
15
20
25
TiB B1 B2 B3 B4 TiC C1 C2 TiD D1 D2 TiE E1 E2
I
n
h
i
b
i
t
i
o
n

z
o
n
e

d
i
a
m
e
t
e
r

(
m
m
)
Pseudo T Pseudo E. coli T E. coli Sal T Sal Proteus


Figure 1. Comparative susceptibility pattern of gram negative test bacterial strains to chromatographic fractions and re-fraction.



plant materials (Anderson, et al., 2001; Dnyaneshwar et
al., 2003). The phytochemicals detected in the crude and
fractions such as flavonoids, tannins, glycosides and
saponins have been associated with antimicrobial activity
by other workers (Marjorie, 1999; Mahajan

and Badgujar,
2008). However, since there is a family of these
compounds, there may be a need to determine which
specific compound(s) amongst them exhibits the
antimicrobial activity. Among the six fractions got initially
from the parent crude ethanolic extract of the stem bark,
TiF showed the least antibacterial activity. With those that
showed better antibacterial activity, the range and type of
organisms showing susceptibility varied with fraction,
which indicates that there were several types of
compounds with antibacterial activity among the phyto-
chemical constituents of the stem bark of the T. indica
plant. Thus, TiB probably contained the highest propor-
tions of these antibacterial compounds followed by TiC,
TiA, TiD and TiE, in that order. It is not surprising,
therefore, that TiB showed a wider spectrum of activity
than other fractions; but whether this superiority of activity
means that it contains more potent antimicrobial
principles or it contains compounds acting together
synergistically or additively needs to be ascertained. On
the other hand, the superior activity could be due to the
presence of higher concentrations of the bioactive
components because of the dose response curve, which
showed that the higher the concentration of extract, the
greater the inhibition zone diameter (U. U. Nwodo and C.
U. Iroegbu, unpublished observation). The same caution
is exercised in the interpretation of the observed greater
proportion of the organisms susceptible to fraction TiA.
Fractionation and re-fractionation in some cases resulted
in improved activity but in others resulted in loss of
activity. For example, when TiB was re-fractionated, one
of its sub-fractions, TiB4, showed no activity against type
P. aeruginosa strains and TiB3 showed no activity
against clinical strain of P. aeruginosa unlike the parent
fraction TiB. Similarly TiB1, TiB2, TiB3 and TiB4 lost
activity against E. coli ATCC 11775; only TiB3 had activity
against E. coli (clin.) in contrast to the parent TiB. These
represent a situation where purification leads to loss of
activity suggesting that components of TiB may have
acted synergistically or additively to produce the activity
observed with the parent fraction. However, TiB3s
activity against E. coli (clin.) indicates that it is probably a
singly active constituent. Conversely, TiC1 exhibited
activity against Salmonella sp. in contrast to the parent
fraction, TiC and sister sub-fraction TiC2 which showed
no activity against this strain. It is yet to be ascertained
that this is a case of TiC2 antagonizing TiC1 in the parent
TIC, thus hindering its activity against the strain. All these
show that purification of crude extracts could produce
loss or gain of activity depending on the nature of
interaction (antagonism or synergism/additivity) between
the constituent compounds of the extract.
Nwodo et al. 7113



0
5
10
15
20
25
TiB B1 B2 B3 B4 TiC C1 C2 TiD D1 D2 TiE E1 E2
I
n
h
i
b
i
t
i
o
n

z
o
n
e

d
i
a
m
e
t
e
r

(
m
m
)
Staph T Staph B. sub B. cereus4 B. cereus5


Figure 2. Comparative susceptibility pattern of Gram positive test bacterial strains to chromatographic fractions and re-fraction.



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